Halogenated Rocaglate Derivatives: Pan-antiviral Agents against Hepatitis E Virus and Emerging Viruses

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4′, 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.


■ INTRODUCTION
−3 They are found in several tree species of the genus Aglaia (Meliaceae) that grow in subtropical and tropical forests of Southeast Asia, Northern Australia and the Pacific region. 4he first rocaglate extracts collected revealed significant activity against P-388 lymphatic leukemia in CDF1 mice and inhibitory activity in vitro against cells derived of human epidermoid carcinoma of the nasopharynx (κB cells).The antileukemic effect was attributed to the 1H-cyclopenta[b]benzofurans rocagloic acid (1a, Figure 1) and rocaglamide (1b). 5Later, antiviral properties against the Newcastle disease virus (NDV) were reported 6 and the biological target of flavaglines was studied for the natural product silvestrol (2a) and 1-O-formylglafoline (1d).The excellent broadband antiviral activity of silvestrol (2a) was substantiated for highly pathogenic Ebola virus, 7 as well as Zika virus, Hepatitis E virus (HEV) and viruses from the Coronaviridae and Picornaviridae family without pronouced cytotoxic effects for immortalized cell lines (Huh-7 and MRC-5). 8Translation initiation is a key process in viral proliferation.Because RNA viruses do not encode their own translational machinery, they rely on host protein synthesis.In the past, targeting the translation machinery of the host has been extensively studied and proposed as a therapeutic strategy for the treatment of viral infections.It is widely accepted that rocaglates exert their biological activity by stimulation of eIF4Af-RNA clamping. 9he eukaryotic initiation factor 4a (eIF4A) is an ATPdependent RNA helicase, responsible for unwinding the secondary structure of mRNAs.Flavaglines force an engagement between eIF4A and RNA that prevents eIF4A from participating in the ribosome-recruitment step of translation.Recently, Iwasaki and co-workers resolved the structure of the human complex composed of eIF4A1, AMPPNP, rocaglamide 1b and polypurine RNA, providing the molecular basis of rocaglamide RNA sequence selectivity.From these X-ray studies it was found that in particular the dimethoxysubstituted aromatic ring A in 1b is directed toward the polypurine RNA.As such, ring A is stacked with the adenine base of A7 and guanine base of G8 nearly in parallel. 10−13 It was precisely this promising biological potential of rocaglates that triggered synthetic programs culminating in the first total synthesis by Trost et al. in 1990 14 and follow-up synthetic programs by the groups of Deśaubry, 15−17 Porco,18,19 Tremblay, 20 Burns, 21 Ishibashi 22 and Reich 23 that provided rocaglate-derived compound libraries.
The majority of these studies primarily focused on the substitution of the methoxy groups at C6 and C4′ and variation of the amide moiety.Both showed a profound effect on biological activity.Unsurprisingly, several halogenated rocaglates were also part of these libraries, as halogens are of great importance in medicinal chemistry.They give, in most cases, advantages to biophysical and -chemical properties of related compounds.Halogen substitution can enhance metabolic stability, lipophilicity and electronegativity.4][15][16]20,27,28 However, the possible impact of the small and highly electronegative fluorine atom as a substituent at C6 or C4′ is so far unknown. Furthermre, no derivatives halogenated at the C8 position have been reported to date.
Consequently, we initiated a program to synthesize and biologically evaluate a library of so far unknown halogenated rocaglate derivatives and tested them against several emerging RNA viruses, including HEV, Chikungunya (CHIKV), Rift Valley fever (RVFV) and SARS-CoV-2 viruses.As part of this program, we also aimed to identify the most practical synthetic route among several options for accessing the target derivatives.

■ RESULTS AND DISCUSSION
General Considerations on the Syntheses.To date, the majority of rocaglate syntheses are based on a biomimetic approach starting from 3-hydroxyflavones (flavonol) and cinnamic acid derivatives, first described by Porco and coworkers in 2004. 29This process first involves UV lightmediated [3+2]-cycloaddition via an excited-state intramolecular proton transfer leading to the aglain core.Subsequently, skeletal rearrangements via a ketol shift and anti-selective reduction of the resulting ketone lead to the cyclopenta[b]benzofuran core present in the rocaglates.Excellent substrate selection and high diastereoselectivity for the establishment of the five stereocenters in only three steps are compelling reasons for the superiority of this route.
Surprisingly, synthetic access to the required 5,7,4′substituted flavonols still poses a major challenge.In previous studies on flavaglines, the flavonols were most commonly prepared via an Algar−Flynn−Oyamada (AFO) reaction 14,22 or alternatively a Baker−Venkataraman synthesis. 20,22,30e first route represents an oxidative cyclization of the corresponding chalcone with NaOH, KOH or K 2 CO 3 in combination with hydrogen peroxide (Scheme 1, Route A).
Although this biomimetic approach allows for rapid access to flavonols, its substrate scope is however rather restricted.In particular, electron-donating substituents at C5 and C7 or electron-withdrawing substituents at C4′ favor the formation of the corresponding aurone instead of the flavonoid. 31,32It should be noted, however, that in principle an alternative type of cyclization to the aurone skeleton is conceivable and possible.
The Baker−Venkataraman synthesis (Scheme 1, Route B) 20 requires a larger number of steps but is supposedly more versatile with respect to substrate scope, as the different electronic properties of the substituents at C5 and C7 have little effect on the formation of flavonol.
The synthesis commenced from the corresponding ohydroxyl acetophenones.A Rubottom oxidation sequence leads to the α-hydroxyacetophenones from which the bisbenzoates are formed by esterification.Depending on the desired substitution pattern on the B ring, various benzoic acid or benzoyl chloride derivatives can be used. 20,22Next, the sequence proceeds through a base-mediated Baker−Venkataraman rearrangement, followed by acid-catalyzed condensation and saponification of the enol ester that yields the flavonol.However, the aforementioned reaction sequence involves harsh basic and acidic conditions, which can limit the application of some protecting and functional groups.
Synthesis of Rocaglates Based on the Baker− Venkataraman Rearrangement.To investigate the influence of halogen substituents at C4′, we resorted to the Baker− Venkataraman route, since the electron-withdrawing effect of fluorine, chlorine and bromine in the AFO reaction strongly favors the formation of aurone.Based on studies by Tremblay et al., 20 we established a reliable, high-yielding and scalable linear route (Scheme 2) where acetophenones 3a and 3b served as starting materials (see the Supporting Information).
Rubottom oxidation and formation of the α-hydroxyacetophenones 4, followed by double esterification with various 4substituted benzoyl chlorides, furnished precursors 5 that are Scheme 1. Synthetic Approaches to Rocaglate Derivatives with 5,7,4′-Substituted Flavonols as Key Intermediates and Structure of Aurones Journal of Medicinal Chemistry required for the Baker−Venkataraman rearrangement, consistently in excellent yields.In the presence of LiHMDS as a base, the anionic rearrangement led to the phenol 6. Next, a ring-closing condensation reaction led to the formation of flavonol esters 7. We found that elevated temperatures were required for substrates with chlorine or bromine substitution at C4′, while complete conversion was already observed at room temperature (rt) for substrates that bear a methoxy or fluorine substituent at this position.Subsequent saponification with sodium hydroxide gave the corresponding flavonols 8a−bc in excellent yields. 33s mentioned before, these harsh acidic/basic reaction conditions were accompanied by several limitations.Incorporation of acid-labile protecting groups like MOM on the phenol functionality, as well as flavonols with sensitive structural modifications on the B-ring such as the pyridine

Journal of Medicinal Chemistry
ring as well as electron-withdrawing groups such as 4nitrobenzene, is not feasible.
With the flavonols in hand, using methyl cinnamate, the synthesis proceeded with a UV light-mediated [3+2]-cycloaddition, followed by a ketol shift and finally diastereoselective reduction of the ketone according to the protocol of Rizzacassa et al. 34 Methyl rocaglates 9a−bc were obtained in good yields.In the cases where a benzyloxy group was installed at C6, we were able to convert it to the corresponding methoxy ethers 11ba−bc via deprotection with H 2 , Pd/C and methylation with trimethylsilyldiazomethane. 20lavonol Synthesis Based on Algar−Flynn−Oyamada-Type Reactions.Next, we turned our attention toward the modification of the C6 and C8 positions of rocaglates.As mentioned above, the AFO synthesis is a promising approach for the synthesis of flavonols that possess an electronwithdrawing substituent at C5 and C7 (corresponding to C6 and C8 in the corresponding rocaglate) and an electronwithdrawing substituent at C4′. Accordingly, we prepared a series of new halogenated rocaglates via the route depicted in Scheme 3. The acetophenones 3c−i and 3n were prepared from their respective 3,5-substituted phenols by acetylation followed by Fries rearrangement, whereas 3j−m were synthesized from their respective 3,5-dimethoxy halobenzenes by acylation and mono-demethylation (see Supporting Information).
According to a procedure by Sale et al., 35 the acetophenones could be easily converted into chalcones 12c−n in the presence of sodium ethoxide as a base.The subsequent AFO reaction using a mixture of NaOH and H 2 O 2 gave the desired flavonols 8c−n in acceptable yields.Remarkably, this protocol also allowed the synthesis of flavonols 8db and 8nb bearing electron-withdrawing substituents at the C4′ position.However, in these cases, significant proportions of corresponding aurones (see Scheme 1) were also formed.Analogous to flavonols 8a−bc prepared via the Baker−Venkataraman route, compounds 8c−n were converted to rocaglate derivatives 9c− n using the established sequence.With the exception of the 4′bromo rocaglates 9db and 9nb, yields of about 50% over three steps were obtained for the major endo-diastereomer.
Conversion of Rocaglate Methyl Esters to the Corresponding Amides.Starting from the new rocaglate methyl esters, selected members of this library were converted into amides (Scheme 4).It was previously demonstrated that the incorporation of both an N,N-dimethylamide and an Nmethoxyamide group can result in significantly improved antiviral activity. 14,23iological Studies.In total, we prepared 33 rocaglates as racemic mixtures via two different routes, with 30 of the derivatives containing one or more halogen atoms.Since it is known from previous work that the presence of a benzyloxy group at position 6 leads to decreased translational inhibition, 21 compounds 9ba, 9bb and 9bc were excluded from the study of antiviral activity.In addition to the resynthesized (±)-rocaglamide (rac-1b), (±)-CR-31-B (rac-1c) and (±)-methylrocaglate (11bc), commercial (−)-silvestrol (2a) also served as a reference compound.
Hepatitis E viruses are characterized by a highly structured 5′ untranslated region (5′ UTR) and rely on cap-dependent translation for their efficient replication. 36Herein, we assessed structure−activity relationships of our new halogenated rocaglates and their potential as antiviral agents against HEV replication by transfecting hepatoma cells (HepG2) with the HEV-3 replicon p6-Gluc and treating these cells with the compounds listed in Figure 2 in concentrations ranging from 0.15 to 1000 nM (Figure 3A,B).Luciferase activity and MTT assays were conducted to measure HEV RNA replication and cell viability, respectively.The obtained EC 50 , EC 90 , CC 50 and selectivity index (SI) values are summarized in Figure 3C and Table 1.
In accordance with previous findings for non-halogenated compounds, 14,23 an example of chlorinated C2-methyl ester 9a (EC 90 = 105.4nM) showed to be inferior in potency compared to its corresponding dimethylamide 14aa (EC 90 = 101.6nM) and its methoxyamide 14ab (EC 90 = 18.2 nM).To further support this outcome, the same series of derivatives with
The observed improvement in EC 90 values for amides may be attributed by the fact that carbonyl groups of the amide serve as better hydrogen bond donors to Gln195 of eIF4A compared to methyl esters. 17,18Notably, enhanced inhibition of HEV replication was observed in the C4′-bromo methyl ester 11bb (EC 90 = 91.).These observations corresponded to the EC 90 trends Br > Cl > OMe > F and Cl > OMe > F for methyl esters and carbonyl amides, respectively.To further elucidate the influence of halogen functionalization, we examined halogenated rocaglates substituted with Br, Cl and F at positions 6 and 8, or both, concerning their antiviral activity against HEV replication.The C8-bromo methyl ester 9l (EC 90 = 304.7 nM) displayed marginally reduced activity compared to compound 9e (EC 90 = 282.4nM) (C8, C6-bromine substitution).Conversely, the introduction of a bromine atom solely at position C6 in 9m (EC 90 = 30.6nM; CC 50 = 13.8 nM) significantly enhanced both activity and cytotoxicity.A similar trend was observed for chlorine-substituted derivatives (compare 9j [EC 90 = 393.5 nM] with 9da [EC 90 = 725.3nM] and 9k [EC 90 = 45.5 nM]).However, C6-and C8-bromine substitutions generally produced more active compounds than their C6-and C8chlorine counterparts.Also, addition of a bromine atom Fluorine functionalization at position C8 in carbonyl amides 14ha (EC 90 = 758.7 nM) and 14hb (EC 90 = 69.8nM) led to reduced activity compared to non-halogenated amides rac-1b and rac-1c.Intriguingly, the introduction of a fluorine moiety at position C6 in 9c (EC 90 > 1000 nM) and 9i (EC 90 > 1000 nM) completely diminished antiviral activity in hepatoma cells.
Collectively, these findings demonstrate that bromine functionalization yielded the most significant improvement of activities when substituted at position C6 (C6 > C4′ > C8), while chlorine substitutions led to the most potent increase in activity for position C4′ (C4′ > C8).Conversely, fluorine functionalization at C4′ and C8 resulted in reduced antiviral activity and cytotoxicity and entirely abrogated activity when introduced at the C6 position (C8 > C4′ > C6).Based on calculated SI values, two additional trends were observed.First, substitutions on ring A (position C6 and C8) tend to result in improved SI values compared to C4′ or C2 substitutions.Also, derivatives with improved activity were observed to have better SI values than less potent derivatives.
Based on selectivity indices calculated for 48-h treated compounds, we identified 9m and 9da as the most promising rocaglates in our investigation (Figure 3A).Consequently, we evaluated the antiviral efficacy of 9da and 9m against CHIKV, RVF and SARS-CoV-2.Derivative 9k and 14m were not included, due to high structural similarity of 9k to 9m and high toxicity observed for 14m at 48 h.Therefore, we also selected derivative 14f for further analysis.The C6, C8-chlorofunctionalized methyl ester 9da proved to be the least active derivative for all tested viruses (Figure 4A−C, Table 2).Nmethoxyamide 14f exhibited less activity than the C6-bromofunctionalized 9m for Chikungunya virus (CHIKV ) [EC 90 = 20.2nM vs EC 90 = 9.8 nM], Rift Valley fever virus (RVFV) [EC 90 = 113.2nM vs EC 90 = 53.2nM] and SARS-CoV-2 [EC 90 = 339.9nM vs EC 90 = 80.0 nM], while 14f and 9m showed similar activity against HEV.Finally, we evaluated the influence of the cell density on the antiviral activity of exemplified for 9m by comparing the standard protocol cell density to that of a confluent monolayer.As depicted in Figure S1, cell viability improved when cell density was higher.However, at the same time the antiviral response of 9m decreased, which is likely due to the greater number of cells replicating the HEV genome, necessitating a higher dose of the drug to achieve the same reduction of replication (Figure S1).

■ CONCLUSION
One of the most promising targets for inhibition of viral protein synthesis is the eukaryotic initiation factor (eIF) 4F complex (comprised of eIF4A, 4E and 4G).Due to a highly structured viral 5′-untranslated region (5′UTR), a large number of RNA viruses require the DEAD-box RNA helicase activity of eIF4A to unwind the viral genome and to allow for the recruitment and scanning of the 43S-pre-initiation complexes (43S-PIC) during translation initiation. 37Intriguingly, several previous studies have reported that inhibition of the eIF4A complex by rocaglates could prevent replication of different RNA viruses in vitro and in vivo. 38In this study, a library of 27 halogenated derivatives of rocaglamide was synthesized via two different synthetic routes.Subsequent biological evaluation of the modified rocaglate derivatives revealed an potential antiviral effect on hepatitis E (HEV) and moderate antiviral activities against Chikungunya (CHIKV), Rift Valley river virus (RVFV) and SARS-CoV-2 viruses.In addition, the compounds exerted some cytostatic effects, which was reflected by the low to moderate SI values.The biological tests revealed various structure−activity findings about the rocaglates, especially with regard to positions 4′, 6 and 8 (Figure 5A−C).For the 4′ position, an increase in activity of F < OMe < Cl < Br was found.The bromine derivative is thus more active than the rocaglate with the methoxy group found in the natural products.The fluorine derivative, on the other hand, exerts hardly any antiviral activity.For the 6 position the trend is as follows.Here fluorine leads to complete loss of antiviral activity followed by OMe < Cl < Br.Finally, the replacement of the methoxy group in position 8 gave the following relationship: Br ∼ Cl < F < OMe.Replacing the methoxy groups at positions 6 and 8 with two identical substituents results in the following picture: F ≪ MeO ∼ Br < Cl.The antiviral activity of the dichloro derivative 9da is further enhanced when the methoxy group at C4′ is replaced by bromine, as in rocaglate 9db.Finally, it was found that the best halogen combination at positions 6 and 8 is bromine at C6 and chlorine at C8 in rocaglate derivative 9f.
It is remarkable that the medicinal-chemically relevant halogen fluorine shows a negative influence on the antiviral properties of rocaglates, at least in particular at positions 4′ and 6, less so at position 8.
Another trend worth mentioning is the fact that substitutions at the A ring (C6, C8) lead overall to better SI values in terms of activity than modifications at C4′ or at C2 (ester to amide).In general, more antiviral active derivatives show on average a better SI value than derivatives with lower activity.
This study contributes to the elucidation of new structure− activity relationship for a series of antiviral compounds targeting a panel of human pathogenic viruses.We identified compounds 9m and 14f, which are all more potent than the natural product (±)-rocaglamide (rac-1b) and similarly potent as (−)-silvestrol (2a), as potential candidates for further studies.The cytotoxicity of these compounds is comparatively low warranting further explorations.Finally, one may speculate about the special effect of halogen substitution presented in this work.The report by Iwasaki and co-workers 10 on the resolved structure of the human complex composed of eIF4A1, AMPPNP, rocaglamide 1b and polypurine RNA provides insight into this matter, because ring A in 1b, that we modified with halogen substituents, is directed toward the polypurine RNA, specifically the adenine base of A7 and guanine base of G8.Halogen bonding, 39 which resembles the electron density donation-based weak interaction of halogens with Lewis bases, including nucleobases, 40 may provide a rationale for the observations reported here.A telling example is clindamycin, a halogenated ribosome binder that binds into the 50S subunit. 41t contains one chlorine atom that is directed toward the sugar edge of guanosine and forms an interaction with the guanine nitrogen atom. 40articularly, the introduction of bromine at position 6 in ring A leads to improved antiviral properties and this may be associated with halogen bonding toward the adenine base of A7 and guanine base at G8 (Figure 5D).In the future, structural biology studies should provide a deeper understanding of the halogen effect observed here.

■ EXPERIMENTAL SECTION
Chemical Synthesis: General Methods.All experiments involving water-sensitive compounds were carried out in dried glassware under argon or nitrogen.Anhydrous solvents (MeCN, CH 2 Cl 2 , Et 2 O, PhMe) were obtained from a M. Braun MB solvent purification system or commercial solvents were used as supplied.Petroleum ether and dichloromethane were distilled before application and triethylamine was dried over KOH and distilled as well.Commercial reagents were used as supplied.Thin-layer chromatography (TLC) was performed on aluminum-backed plates precoated (0.25 mm) with silica gel 60 F254 with a suitable solvent system and was visualized using UV fluorescence and/or developed with KMnO 4 , anisaldehyde or vanillin stain followed by brief heating.For column chromatography, silica gel (35−70 μm) was used.Alternatively, a Biotage SP purification system was used.Biotage silica cartridges were used as supplied.All compounds are >95% pure.The purity of tested compounds was determined by analytical liquid Designations of R 1 −R 3 and X are presented in Figure 2. chromatography of solutions of the compounds in DMSO-d 6 .Waters Alliance 2695 LC with a Waters Acquity 2996 photodiode array detector equipped with a Varian Polaris C18-A column (5.0 μm, 50 mm × 2.0 mm).The mobile phases were (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile.After injection the gradient holds were at A/B (90%/10%) for 1.00 min followed by a gradient to A/B (0%/100%) over 1.75 min, a 0.05 min flush at 0%/ 100% (A/B) and a 1.20 min re-equilibration at A/B (90%/10%) at a flow rate of 0.8 mL/min and a column temperature of 45 °C. 1 H NMR spectra are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, qi = quintet, sx = sextet, sp = septet, bs = broad singlet, m = multiplet), coupling constant (J) in hertz (Hz), integration and assignment. 13C NMR spectra are represented as follows: chemical shift, substitution (p = primary, s = secondary, t = tertiary, q = quaternary) and assignment. 19F NMR spectra are represented as follows: multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, qi = quintet, sx = sextet, sp = septet, bs = broad singlet, m = multiplet), coupling constant (J) in hertz (Hz), integration and assignment.The numbering of the carbon and hydrogen atoms of the rocaglates synthesized follows the IUPAC nomenclature.A list of all rocaglates including the numbering of the carbon and hydrogen atoms is provided in the Supporting Information.Synthesis of (±)-Methyl (1R,2R,3S,3aR,8bS)-3a-(4-chlorophenyl)-1,8b-dihydroxy-6,8-dimethoxy-3-phenyl-2,3,3a,8btetrahydro-1H-cyclopenta[b]benzofuran-2-carboxylate (9a).

-(Benzyloxy)-2-(4-bromophenyl)-5-methoxy-4-oxo-4H-chromen-3-yl 4-bromobenzoate (7bb).
A suspension of crude phenol 6bb (4.42 g, 6.76 mmol, 1.00 equiv) in AcOH (92.0 mL) was treated with H 2 SO 4 (96 wt%, 2.09 mL, 35.4 mmol, 5.24 equiv) and stirred at 50 °C for 20 h.The reaction mixture was poured into ice-cold H 2 O, the yellow suspension was filtered and the precipitate was washed with H 2 O.The wet solid was suspended in a minimal amount of EtOH and heated under refluxing conditions for 45 min.After cooling to rt, the mixture was filtered, the precipitate was washed with cold EtOH and dried under reduced pressure to give a mixture of 7bb and ∼40% of the debenzylated flavonol ester.The solid was dissolved in DMF (65.0 mL) and treated with BnBr (807 μL, 6.76 mmol, 1.00 equiv) and K 2 CO 3 (1.87 g, 13.5 mmol, 2.00 equiv), stirred at rt for 2.5 h and then diluted with CH 2 Cl 2 (100 mL) and NaCl solution (sat., aq., 100 mL).The phases were separated and the organic phase was dried over MgSO 4 , filtered and concentrated under reduced pressure.

Scheme 2 .
Scheme 2. Synthesis of Rocaglates by the Baker−Venkataraman Route a

Scheme 4 .
Scheme 4. Transformation of Selected Methyl Esters to the Corresponding N,N-Dimethyl and N-Methoxymethyl Amides a

Figure 3 .
Figure 3. Antiviral efficacy of halogenated rocaglates against HEV.A) Schematic representation of assay setup.B) Plate layout for in vitro testing.C) EC 90 , EC 50 , CC 50 and SI values derived from dose−response curves at 24 and 48 h post-electroporation.

Figure 4 .
Figure 4. Pan-antiviral inhibition of HEV, Chikungunya virus (CHIKV), Rift Valley fever virus (RVFV) and SARS-CoV-2 replication by 9m, 9da and 14f.A) HEV subgenomic replicon HEVp6-Gluc was electroporated into HepG2 cells.Cells were treated with 9m, 9da and 14f at concentrations ranging from 0.15 nM to 1000 nM for 24 and 48 h.Depicted are nonlinear fit response curves representative of three biological replicates for HEVp6-Gluc (dark blue lines), and cell viability was monitored by MTT assay (gray lines).Error bars indicate standard deviation, n = 3. B) Huh-7 cells were treated with different concentrations (0.15 nM to 1000 nM) of 9m, 9da and 14f and infected at a MOI of 2.5 with infectious clone CHIKV LR2006-OPY1 expressing GFP under the control of a subgenomic promotor.GFP expression as measure of infection (left panel) and cell viability (right panel) were measured by live cell imaging and MTT assay, respectively.C) Vero-E6 cells were infected with SARS-CoV-2 or RVF strain MP-12 at a multiplicity of infection (MOI) of 0.1.Supernatants were collected at 24 h post infection (hpi) or 48 hpi and subjected to RT-qPCR analysis as measure of infection (left and middle panel).Cell viability was determined by MTT assay (right panel).

Figure 5 .
Figure 5. Short summary of SAR analysis and proposed interaction of bromine substituent at C6 of ring A with the polypurine chain.a − C(O)NMe 2 and −C(O)NHOMe instead of −CO 2 Me ester.b − C(O)NHOMe shows improved activity over −CO 2 Me ester.

Table 1 .
Overview of Halogenated Rocaglates Synthesized in the Present Work and Their Corresponding Efficacy against HEV at 24 and 48 h a therefore do not necessarily represent the ratio between EC 50 and CC 50 values listed in the table.
a Designations of R 1 −R 3 and X Are Presented in Figure2.SI values represent mean SI values calculated from three biological replicates and

Table 2 .
Overview of Halogenated Rocaglates Synthesized in the Present Work and Their Corresponding Efficacy against Chikungunya Virus (CHIKV), SARS-CoV-2 and Rift Valley Fever Virus (RVFV) at 24 and 48 h, Respectively a CC 50 and warmed up to rt.The layers were separated and the aqueous layers were extracted with CH 2 Cl 2 (3 × 50 mL).The collected organic layers were washed with brine, dried over MgSO 4 , filtered and concentrated in vacuo.The crude/biphasic solution was diluted with Et 2 O, washed with a saturated aqueous NH 4 Cl solution, dried over MgSO 4 , filtered and concentrated in vacuo.The solvent residue was removed under high vacuum and the crude TBS-enol ether as thick red syrup was used directly for the next step.A suspension of NaHCO 3 (3.21g, 38.2 mmol, 2.50 equiv) and mCPBA (77 wt%, 6.04 g, 35.0 mmol, 1.60 equiv) in dry CH 2 Cl 2 (44 mL) was prepared and stirred at rt for 30 min.A solution of crude TBS-enol ether (6.50 g) in dry CH 2 Cl 2 LiHMDS (1 M in THF 10.4 mL, 10.4 mmol, 3.00 equiv) was added to the crude extract 5ba (3.47 mmol) in THF (19 mL) at −30 °C and stirred at the same temperature for 1.5 h.The reaction was terminated by the addition of a saturated aqueous NH 4 Cl solution at −30 °C and warmed up to rt.The mixture was extracted with EtOAc (3 × 50 mL).The organic layers were washed with water and NaCl solution (sat., aq., 100 mL), dried over MgSO 4 , filtered and concentrated in vacuo.